High Quantity Of DNA Research Articles

Abstract 2292: Impact of delayed processing on cfDNA quantity and quality using STRECK cfDNA tubes: Connect Pilot Study

Abstract Interest in cell-free DNA detection for multi-cancer early detection and tumor monitoring is growing.1 Prospective evaluations of liquid biopsy assays are critical, and knowledge gaps in standard collection and preparation practices must be addressed for adequate assay execution.2 The NCI Connect for Cancer Prevention Cohort Study (Connect) aims to study new approaches in early detection. A pilot study was conducted with the STRECK Cell-Free DNA BCT tube to determine the impact of processing time on cfDNA quantity and quality utilizing two complementary quality control assays. 4 STRECK DNA BCT tubes were drawn from 40 donors. Plasma samples were analyzed, with half processed immediately and the other half stored for five days before processing. Two QC assays were performed to compare results at the two processing time points. The Alu EFIRM assay measured Alu element concentration (copies/mL). The Cell-free DNA ScreenTape assay evaluated Qubit DNA concentration (μg/mL) and %cfDNA. Descriptive statistics and paired sample t-tests were performed to compare mean concentrations at the two processing time points, with and without identified samples with cytolysis. Median, mean, and range were recorded for each assay measure (Table 1). A reduction in cfDNA quantity was observed at the 5-day processing time point. Five 5-day processing samples were identified with high DNA quantity and a high Alu element concentration. The five samples experienced hemolysis and clotting as a result of handling and processing at odds with standard protocol. There was a significant difference between the mean DNA quantities of the processing time points following exclusion of the five samples. There was no change in significance between the mean Alu concentration and %cfDNA following exclusion (Table 2). Both assays successfully detected samples with high nuclear DNA, a major contaminant for cfDNA assays, and identified a reduction in cfDNA at the 5-day processing time point. Proper handling and processing procedures are necessary to avoid cell lysis and maximize cfDNA quality and yield. This pilot establishes a starting point for future evaluations of liquid biopsy tube types, processing delays, and preanalytical cfDNA quality variables. Citation Format: Jazmyn Bess, Domonique Hopkins, Norma Diaz Mayoral, Heather Cooley, Angela de Palatis, David T. Wong, Stephanie Weinstein Oshinsky, Christian Abnet, Marie-Josephe Horner, Amanda Black, Michelle Brotzman, Kathleen Wyatt, Lynn Sorbara, Matthew Young, Mia Gaudet, Nicolas Wentzensen, Mickey Williams, Christopher Karlovich, Eric Greenbank, David Elashoff, Fang Wei, Feng Li. Impact of delayed processing on cfDNA quantity and quality using STRECK cfDNA tubes: Connect Pilot Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2292.

Introducing a Rapid DNA Analysis Procedure for Crime Scene Samples Outside of the Laboratory-A Field Experiment.

Technological innovations enable rapid DNA analysis implementation possibilities. Concordantly, rapid DNA devices are being used in practice. However, the effects of implementing rapid DNA technologies in the crime scene investigation procedure have only been evaluated to a limited extent. In this study a field experiment was set up comparing 47 real crime scene cases following a rapid DNA analysis procedure outside of the laboratory (decentral), with 50 cases following the regular DNA analysis procedure at the forensic laboratory. The impact on duration of the investigative process, and on the quality of the analyzed trace results (97 blood and 38 saliva traces) was measured. The results of the study show that the duration of the investigation process has been significantly reduced in cases where the decentral rapid DNA procedure was deployed, compared to cases where the regular procedure was used. Most of the delay in the regular process lies in the procedural steps during the police investigation, not in the DNA analysis, which highlights the importance of an effective work process and having sufficient capacity available. This study also shows that rapid DNA techniques are less sensitive than regular DNA analysis equipment. The device used in this study was only to a limited extent suitable for the analysis of saliva traces secured at the crime scene and can mainly be used for the analysis of visible blood traces with an expected high DNA quantity of a single donor.

DNA release from plant tissue using focused ultrasound extraction (FUSE).

Sample preparation in genomics is a critical step that is often overlooked in molecular workflows and impacts the success of downstream genetic applications. This study explores the use of a recently developed focused ultrasound extraction (FUSE) technique to enable the rapid release of DNA from plant tissues for genetic analysis. FUSE generates a dense acoustic cavitation bubble cloud that pulverizes targeted tissue into acellular debris. This technique was applied to leaf samples of American chestnut (Castanea dentata), tulip poplar (Liriodendron tulipifera), red maple (Acer rubrum), and chestnut oak (Quercus montana). We observed that FUSE can extract high quantities of DNA in 9-15 min, compared to the 30 min required for control DNA extraction methods. FUSE extracted DNA quantities of 24.33 ± 6.51 ng/mg and 35.32 ± 9.21 ng/mg from American chestnut and red maple, respectively, while control methods yielded 6.22 ± 0.87 ng/mg and 11.51 ± 1.95 ng/mg, respectively. The quality of the DNA released by FUSE allowed for successful amplification and next-generation sequencing. These results indicate that FUSE can improve DNA extraction efficiency for leaf tissues. Continued development of this technology aims to adapt to field-deployable systems to increase the cataloging of genetic biodiversity, particularly in low-resource biodiversity hotspots.

Effect of storage temperature and duration on direct PCR amplification of various feather types and DBS matrices

The use of direct PCR has been pioneered over the last decade for DNA analysis of biological specimens of distinct origins. The information on how longer these specimens can be stored and amplified by direct PCR is however scanty. Such a piece of information could expedite research and diagnostic studies without compromising the reliability of results. The current study was therefore designed to analyze the effect of storage temperature and duration on direct PCR amplification of biological specimens having either low quantity or high quantity of DNA. Whole blood, dried blood spots (DBS), and feathers from chicken were stored for five years at three different temperatures, viz. room temperature (∼25 °C), 4 °C, and −20 °C. These samples were subjected to crude DNA extraction by diluting them in PBS buffer and heating at 98 °C after 1 day, 7 days, 15 days, 1 month, 3 months, 6 months, 1 year, 3 years and 5 years of storage. The crude DNA was PCR-amplified with the use of DNA sexing primers as well as DNA barcoding primers. Incubation at 98 °C for 10 min of any type of sample in PBS buffer was sufficient for crude DNA extraction. There was irrelevant impact of feather type, DBS matrix nature and storage temperature on amplification success over the period of analysis. It was possible to successfully accomplish the amplification of 96 samples with the use of routine PCR reagents within 3.5–6.0 hrs. In short, economical and fast genetic analysis of commonly used avian samples is feasible after their storage for longer time at room temperature.

Highly Sensitive Electrochemical Determination of Lead(II) by Double Stranded DNA (dsDNA) with a Carbon Paper/Reduced Graphene Oxide (CP/rGO) Substrate by Differential Pulse Anodic Stripping Voltammetry (DPASV)

Lead pollution may cause serious damage to environment safety and human health, especially to children’s growth. In this work, by using double stranded DNA (dsDNA) as the recognition element, a simple and effective electrochemical DNA sensor has been developed on a full carbon-based substrate, reduced graphene oxide/carbon paper (rGO/CP), to accomplish the rapid, sensitive and selective determination of Pb2+. Carbon paper (CP) provides a suitable substrate for the sensor with high portability and low cost, while rGO is easily electrodeposited onto CP and serves as both the signal amplification element and the molecular bridge between DNA and CP. Because of its high specific surface area and unique conjugated giant π structure, rGO tightly binds with DNA through π-π stacking and hydrophobic interactions, thereby providing loading of high quantities of DNA. The specific coordination between DNA and Pb2+ further improves the sensitivity of the sensor. The results demonstrate that rapid electrochemical enrichment of Pb2+ is achieved using the constructed CP/rGO/DNA electrode with a linear relationship between the peak current and the concentration of Pb2+ from 1 pM to 100 pM and a detection limit of 1 pM. In addition, this approach shows good selectivity and satisfactory repeatability for river water analysis. A promising sensitive, low-cost, simply-fabricated, and portable full carbon-based electrochemical biosensing platform has been therefore provided for lead determination.

Transfer, persistence and recovery of DNA and mRNA vaginal mucosa markers after intimate and social contact with Bayesian network analysis for activity level reporting

In sexual assault cases, it can be challenging to identify the type of body fluids/ cell types present in a crime scene sample, especially the origin of epithelial cells. Therefore, more labs are applying mRNA body fluid analysis for saliva, skin and vaginal mucosa markers. To address activity level propositions, it is necessary to assign probabilities of transfer, persistence, prevalence and recovery of DNA and mRNA markers. In this study we analysed 158 samples (fingernail swabs, penile swabs and boxershorts) from 12 couples collected at different time points post intimate contact and after non-intimate contact in order to detect DNA from the person of interest (POI) and mRNA vaginal mucosa markers. Samples were DNA and RNA co-extracted and analysed with PowerPlex®Fusion 6C System and 19-plex mRNA primer mix respectively, using Endpoint PCR and the CE platform. Vaginal mucosa was detected up to 36 h post intimate contact, but also detected in one non-intimate contact sample. In 94% of intimate contact and 50 % of non-intimate contact samples the DNA results support the proposition that POI is the donor (LR ≥ 10,000). There was a strong association between the detection of vaginal mucosa and the average RFU value of the POI. The data were used to instantiate a comprehensive Bayesian network to evaluate the evidence at activity level, given alternate propositions conditioned upon indirect or direct transfer events. It is shown that the value of the evidence is mainly affected by the high DNA quantity (measured as mean RFU) that is recovered from the POI. The detection of vaginal mucosa had low impact upon the resultant likelihood ratio.

Comparing DNA yield from fish scales following different extraction protocols

Studies on genetic diversity, adaptive potential and fitness of species have become a major tool in conservation biology. These studies require biological material containing a reliable source of DNA which can be extracted and analysed. Recently, non-invasive sampling has become the preferred sampling method of such biological material; particularly when studying endangered species. Elasmoid scales from teleost fish are an example of non-invasive samples from which DNA can successfully be extracted. This study compared different extraction protocols to find an optimal method for extracting DNA from teleost fish scales. This was done with the intent to use the protocol that yielded the highest quantity of DNA on dried, archived scales. The protocols tested in this study included (1) phenol/chloroform with a TNES-urea digestion buffer, (2) phenol/chloroform with an amniocyte digestion buffer and (3) Qiagen DNeasy Blood and Tissue Kit with variations in incubation times and temperatures of each protocol. While the phenol/chloroform with TNES-urea digestion buffer yielded significantly higher concentrations of DNA compared to the other protocols, all protocols followed in this study yielded sufficient quantities of DNA for further downstream applications. Therefore, while there are multiple viable options when selecting a DNA extraction protocol, each research project’s individual needs, requirements and resources need to be carefully considered in order to choose the most effective protocol.

Intra-bone nuclear DNA variability and STR typing success in Second World War first ribs

DNA sampling and typing are used for identifying missing persons or war victims. In recent forensic studies, little focus has been placed on determining intra-bone variability within a single skeletal element. When dealing with aged human bones, complete skeletal remains are rarely present. In cases in which only the torso is available, studies have shown that ribs are one of the most appropriate samples, but intra-bone variability has not yet been studied. A higher degree of remodeling was found to contribute to higher DNA yield in the parts of the skeletal element where the most strain is concentrated. This study explores intra-bone variability in proximal, middle, and distal parts of the first human rib by determining the quantity and quality of DNA using the PowerQuant System (Promega) and autosomal STR typing success using the PowerPlex ESI 17 Fast System (Promega). Thirty first ribs from a single Second World War mass grave were sampled. No variation in DNA degradation was observed across the individual rib. The highest quantity of DNA was measured in the proximal part of the first rib, and in all ribs except three, full or almost full genetic profiles were obtained. Thus, when only the torso is present in archaeological or medico-legal cases, first ribs are recommended to be collected if possible, and the proximal or vertebral ends should be sampled for genetic analysis.

Minimal residual disease (MRD) in patients with resected stage I NSCLC: Results of the prospective adjuvant IFCT-0703 trial.

8526 Background: MRD aims to detect circulating biomarkers of micrometastatic disease and ultimately predict recurrences. The IFCT-0703 randomized phase II trial failed to show a benefit of 6 months adjuvant pazopanib (P) vs. placebo after resection of stage I NSCLC (7th TNM edition). The outcome of pts based on their MRD status has been evaluated. Methods: Blood samples were collected in EDTA tubes (Becton Dickinson Company) after surgery (T0), after 3 months (T3) of P or placebo and at the end of treatment (T6). Plasmas were obtained after double centrifugation of total blood. Total nucleic acid was extracted using the Maxwell RSC LV plasma kit (Promega) according to the manufacturer’s protocol. Samples were quantified using the QuBit dsDNA HS Assay kit on a QuBit 3.0 flurometer (Thermo Fisher Scientific). Molecular analysis was performed by next generation sequencing using the Oncomine Lung cfDNA Assay (ThermoFisher Scientific). Two MRD definitions were tested : 1) high level of DNA in the blood or 2) any mutation detected by the standard bioinformatic pipeline was considered present, whatever the allelic fraction. Results: 143 pts were randomized in 29 centers between March 2009 and August 2012, 71 and 72 in the placebo and P arms respectively. Among the 119 pts with evaluable T0 samples, 27 pts recurred and 14 died. Median DNA concentration ([DNA]) was 6.6 ng/ml and an increase of [DNA] of 10 ng/ml was found prognostic of poor DFS and OS, HR=1.4, 95%CI [1.14-1.72], p=0.0016 and HR=1.62, 95%CI [1.15-2.30], p=0.0057 respectively. In 81 pts with available T0-T6 samples, [DNA] variation had no different impact on DFS and OS, in the P arm and the placebo arm. ctDNA mutations (ctDNA+) were detected in 31/119 pts. ctDNA+ were more frequent in samples with high DNA quantity (p=0.0002). Genes mutated at T0 were TP53 in 16, NRAS in 6, MAP2K1 in 2, KRAS in 1, EGFR in 5, BRAF in 1, ALK in 2. 29 pts had 1 mutation, 2 had 2 mutations. DFS and OS were similar between pts with or without ctDNA+ : HR= 1.038 (95%CI 0.438-2.456, p=0.93) and 1.193 (95% CI 0.367-3.882, p=0.77) respectively. Among 27 pts with ctDNA+ at T0 and available sample at T6, 23 had no more mutations at T6. Two pts had a ctDNA+ only at T6 (not at T3), one of them had a recurrence at 7 months. Conclusions: Post-operative ctDNA mutations are found in 26.0% of the pts but their positivity had no impact on DFS or OS. In contrast, DFS and OS were poorer in pts with increased plasma DNA concentration. ctDNA mutations status do not recapitulate the complexity of MRD characterization. NGS will be performed on matched tissues in order to refine MRD definition. Clinical trial information: NCT00775307.

Electrospun PLGA Fiber Diameter and Alignment of Tendon Biomimetic Fleece Potentiate Tenogenic Differentiation and Immunomodulatory Function of Amniotic Epithelial Stem Cells.

Injured tendons are challenging in their regeneration; thus, tissue engineering represents a promising solution. This research tests the hypothesis that the response of amniotic epithelial stem cells (AECs) can be modulated by fiber diameter size of tendon biomimetic fleeces. Particularly, the effect of electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned microfibers possessing two different diameter sizes (1.27 and 2.5 µm: ha1- and ha2-PLGA, respectively) was tested on the ability of AECs to differentiate towards the tenogenic lineage by analyzing tendon related markers (Collagen type I: COL1 protein and mRNA Scleraxis: SCX, Tenomodulin: TNMD and COL1 gene expressions) and to modulate their immunomodulatory properties by investigating the pro- (IL-6 and IL-12) and anti- (IL-4 and IL-10) inflammatory cytokines. It was observed that fiber alignment and not fiber size influenced cell morphology determining the morphological change of AECs from cuboidal to fusiform tenocyte-like shape. Instead, fleece mechanical properties, cell proliferation, tenogenic differentiation, and immunomodulation were regulated by changing the ha-PLGA microfiber diameter size. Specifically, higher DNA quantity and better penetration within the fleece were found on ha2-PLGA, while ha1-PLGA fleeces with small fiber diameter size had better mechanical features and were more effective on AECs trans-differentiation towards the tenogenic lineage by significantly translating more efficiently SCX into the downstream effector TNMD. Moreover, the fiber diameter of 1.27 µm induced higher expression of pro-regenerative, anti-inflammatory interleukins mRNA expression (IL-4 and IL-10) with favorable IL-12/IL-10 ratio with respect to the fiber diameter of 2.5 µm. The obtained results demonstrate that fiber diameter is a key factor to be considered when designing tendon biomimetic fleece for tissue repair and provide new insights into the importance of controlling matrix parameters in enhancing cell differentiation and immunomodulation either for the cells functionalized within or for the transplanted host tissue.

Determination of fusarium crown rot resistance in wheat to Fusarium culmorum and Fusarium pseudogramineaum using real time PCR

Wheat varieties from Turkey, Australia and the Pacific Northwest of the US were screened for resistance to Fusarium crown rot (Fusarium pseudograminearum and Fusarium culmorum) in inoculated growth chamber studies. Fungal DNA was also quantified from the lower stems (crowns) of seedlings using quantitative PCR and crown rot scores were correlated with DNA results. There were significant differences among the varieties for crown rot severity index (p = 0.0265), crown rot score (p = 0.0003), disease index on leaf sheaths (leaf sheath score) (p = 0.0013), lesion length (p = 0.0200), and plant height (p < 0.0001). The crown rot score and plant height measurements also showed that the isolate of F. culmorum was more virulent than the isolate of F. pseudograminearum. The correlations were strongest between crown rot score and DNA levels at the second harvest, and much stronger with the isolate of F. culmorum compared to the isolate of F. pseudograminearum. Some susceptible varieties such as Seri had both a high disease score and high quantities of DNA, with both F. culmorum and F. pseudograminearum. On the other hand, varieties such as Matt, Sunco and Otis showed intermediate to high crown rot scores in response to F. culmorum, but much lower levels of DNA than Seri. 2–49, Burbot-6 and CT000161, in response to F. culmorum, showed low levels of disease and low levels of DNA. In conclusion, there were examples of varieties that were resistant and tolerant (low levels of DNA and disease), resistant and intolerant (low quantities of DNA, but higher levels of disease) and susceptible and intolerant (high quantities of DNA and disease). Quantitative PCR in combination with classical phenotyping of resistance to Fusarium crown rot may provide a tool for distinguishing resistance and tolerance in breeding efforts.

Determination of suitable insect part for non‐lethal DNA sampling: case study of DNA quality and regeneration capability of dragonflies

Abstract Genetics has been widely used in insect ecology and conservation. To minimise the effect of DNA sampling on organisms as much as possible, it would be ideal to use non‐invasive or non‐lethal DNA sources. Therefore, it is also very important to determine the responses of organisms to DNA sampling. In this study, the quality and quantity of genomic DNA samples from three types of insect tissues (exuvia, mid‐legs and wings) were evaluated. As model organisms, we used two dragonfly species of different sizes (Leucorrhinia dubia, Anax imperator). We also tested the regenerative ability of dragonfly larvae as a repair mechanism after mid‐leg cut‐off, with respect to factors such as size and quantity of diet. We found that DNA of sufficient quality for analyses was obtained from all tested tissues. Nonetheless, isolates from exuviae were conclusively less useful for sequencing than those from the mid‐legs and wings. The highest quantity of DNA was obtained from the mid‐legs. The survival of larvae is not affected by removing the legs, which can usually regenerate. All the tested tissues could be a source of adequate DNA; however, we concluded that primarily the legs should be used because they provided the best DNA samples in terms of quantity and quality of DNA. Furthermore, their exploitation would not affect individuals seriously if young larvae with sufficient time (at least 6 months) for regeneration are sampled. The exuviae should be used for absolutely non‐invasive studies involving endangered or protected species.

Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics.

Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.

A new set of DIP-SNP markers for detection of unbalanced and degraded DNA mixtures.

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP-SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP-SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP-SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP-SNPs in a Chinese Han population. The DIP-SNPs were capable of detecting the minor contributor's allele in home-made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1-10ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP-SNPs may serve as a valuable tool in detection of UDM in forensic medicine.

Characterization of mucoid and serous middle ear effusions from patients with chronic otitis media: implication of different biological mechanisms?

BackgroundChronic Otitis Media with effusion (COME) is characterized by persistent middle ear effusions that are in most cases highly viscous, but some patients present with serous fluid. This study aimed at comprehensively characterizing the macromolecular composition of mucoid vs. serous MEEs.MethodsMEEs from patients with COME were analyzed for proteins by Mass Spectrometry (MS) and western blot techniques, total DNA quantity, bacterial DNA (16S sequencing) and cytokine content. Proteomics datasets were studied in Ingenuity pathway analysis (IPA).ResultsMucoid samples showed a global tendency of increased pro- inflammatory mediators. IL1-β and IL-10 were significantly more abundant in serous samples (p<0.01). Mucoid samples had higher DNA quantity (p=0.04), more likely to be positive in MUC5B protein (p=0.008) and higher peptide counts (12786 vs 2225), as well as an overall larger number of identified proteins (331 vs 177), compared to serous. IPA found the mucoid sample dataset to be related to immune cell function and epithelial remodeling whereas the serous sample dataset showed acute responses and blood related proteins. Interestingly, serous samples showed more bacterial DNA than mucoid ones, with less bacterial genera variability.ConclusionThis study demonstrates divergent immune responses in children with COME by effusion quality.

OPTIMATION OF DNA EXTRACTION PROTOCOL OF Elaeidobius kamerunicus

African pollination weevil (Elaeidobius kamerunicus Faust) has an important role in the productivity of Indonesian oil palm plantation. Up to now, there has not been a comprehensive biological study of the species at molecular level. The basic knowledge is very useful for exploitation of the weevil for effective oil palm fruit set development. This research aimed to obtain DNA extraction protocol of E. kamerunicus for DNA fingerprinting of the species. Results showed that using a DNA extraction kit,material disruption by using micro pestle resulted the highest quantity of DNA, while there were no significant differences of resulted DNA quantity among treatments using tissue lyser for material disruption. DNA extracted by using micro pestle or tissue lyser for material disruption is adequate for DNA fingerprinting using AFLP (Amplified Fragment Length Polymorphism) and sequencing techniques.

Augmentation of wedelolactone through in vitro tetraploid induction in Eclipta alba (L.) Hassk.

A complete protocol for the in vitro induction of Eclipta alba tetraploids has been optimized to enhance the wedelolactone content, an anti-cancerous compound. The effects of different concentrations of colchicine (0, 0.01, 0.05, 0.1, 0.2 and 0.3%; w/v) along with treatment durations (12, 24, 36 and 48 h) were investigated on shoot tip (ST) and nodal segment (NS). The treated explants were then incubated on Murashige and Skoog (MS) medium having 1.5 mg L−1 N6-benzylaminopurine and 0.5 mg L−1 α-napthalene acetic acid for shoot regeneration and afterward root was induced on 1.0 mg L−1 indole-3-acetic acid enriched ½MS medium. The tetraploids of E. alba were proficiently induced by the treatment of 0.1% colchicine for 24 h. The highest tetraploid induction efficiency was obtained from ST (30.56%) in comparison to the NS (22.22%). Analysis by spectrophotometry and flow cytometry showed that colchicine treated plants contained higher quantity of DNA than diploid plants. Cytological studies demonstrated doubled the chromosome number in tetraploids (2n = 4x = 44) than diploids (2n = 2x = 22). The ploidy level enhancement lead to alteration of other traits, like increased plant height, stem diameter, leaf size, stomatal size and chlorophyll content. As determined through high performance thin-layer chromatography, the ultimate achievement of this technique is the higher accumulation of wedelolactone in tetraploid plants (300.32 µg g−1 dry weight) in evaluation to in vitro diploid (131.31 µg g−1 dry weight) and in vivo diploid mother plants (93.26 µg g−1 dry weight), thus improving the pharmaceutical value of E. alba.

Abstract B39: A pilot study to establish procedures for DNA and RNA isolation from African esophageal tumor specimens

Abstract Objective: We propose to utilize tumor biospecimens from patients with esophageal squamous cell carcinoma (ESCC) to identify possible genetic, molecular, and infectious determinants of this high-incidence disease in East Africa. However, laboratory technologies in East Africa are not available to support DNA and RNA extraction and genome and transcriptome sequencing from patient specimens. In preparation for establishment of a biorepository for African ESCC specimens, we compared different fixation and preservation media for transportability over long distances and success in preserving the genetic integrity and expression profiles of ESCC. Methods: Patients with a suspected diagnosis of ESCC at Muhimbili National Hospital in Dar es Salaam, Tanzania were identified and consented prior to endoscopic evaluations. For patients with endoscopic findings consistent with ESCC, tumor biopsies were obtained and stored using two different fixation and preservation media: PAXgene® Tissue Container (n=2) and RNAlater® (n=2). All specimens were shipped at room temperature from Dar es Salaam to San Francisco. DNA and RNA quantity was measured by nanodrop method and DNA was further confirmed by picogreen method, yielding measures of total DNA and RNA acquired (ug). DNA quality was measured as percent of total DNA degradation to between 200-1000 bp. RNA quality measure was determined by bioanalyzer output measure RNA Integrity Number (RIN). Results: Specimens for our first 10 patients were analyzed. Average transit time of biopsy specimens in preservative at room temperature was 6.2 days (range 3.5-9.0). Tumor specimens preserved with PAXgene® yielded a mean of 7.7 ug total DNA and 8.6 ug total RNA. Tumor specimens preserved with RNAlater® yielded a mean 1.4 ug of DNA and 8.2 ug of RNA. DNA degradation products were 0% of total with PAXgene® versus 6% of total with RNAlater®. Specimens preserved with PAXgene® yielded a mean RIN of 4, while RNAlater® yielded a mean RIN of 9. DNA and RNA quality were not associated with length of time at room temperature, up to a maximum of 9 days. Conclusion: Tissue preserved using the PAXgene® Tissue Container yielded higher DNA quantity and quality than tissue preserved in RNAlater®. RNA quantity was comparable for both mediums, but RNAlater® resulted in superior RNA quality versus PAXgene®. Both mediums allowed for flexible transport of specimens at room temperature and merit further inquiry into their potential as cost-effective methods to facilitate molecular analyses for geographically isolated diseases in Africa. Based upon our pilot data, each medium offers unique advantages. Our expanded study will continue to utilize both mediums to optimize isolation of both DNA and RNA. We will plan to present data for an enriched sample size. Funding source: National Institutes of Health, National Cancer Institute Cancer Center Administrative Supplement to Promote Cancer Prevention and Control Research in Low and Middle Income Countries, A119617, [CA-0082629]. Citation Format: Beatrice Paul Mushi, John Greer, Charles William Cahalane, II, Msiba Selekwa, Ali Mwanga, Larry Akoko, Elia Mmbaga, Eric Collisson, Katherine Van Loon. A pilot study to establish procedures for DNA and RNA isolation from African esophageal tumor specimens [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B39.

Comparison of four DNA extraction methods for comprehensive assessment of 16S rRNA bacterial diversity in marine biofilms using high-throughput sequencing

High-throughput DNA sequencing technologies are increasingly used for the metagenomic characterisation of microbial biodiversity. However, basic issues, such as the choice of an appropriate DNA extraction method, are still not resolved for non-model microbial communities. This study evaluates four commonly used DNA extraction methods for marine periphyton biofilms in terms of DNA yield, efficiency, purity, integrity and resulting 16S rRNA bacterial diversity. Among the tested methods, the Plant DNAzol® Reagent (PlantDNAzol) and the FastDNA® SPIN Kit for Soil (FastDNA Soil) methods were best suited to extract high quantities of DNA (77-130 μg g wet wt-1). Lower amounts of DNA were obtained (<37 μg g wet wt-1) with the Power Plant® Pro DNA Isolation Kit (PowerPlant) and the Power Biofilm® DNA Isolation Kit (PowerBiofilm) methods, but integrity and purity of the extracted DNA were higher. Results from 16S rRNA amplicon sequencing demonstrate that the choice of a DNA extraction method significantly influences the bacterial community profiles generated. A higher number of bacterial OTUs were detected when DNA was extracted with the PowerBiofilm and the PlantDNAzol methods. Overall, this study demonstrates the potential bias in metagenomic diversity estimates associated with different DNA extraction methods.

Novel APC gene mutations associated with protein alteration in diffuse type gastric cancer

BackgroundThe role of adenomatous polyposis coli (APC) gene in mitosis might be critical for regulation of genomic stability and chromosome segregation. APC gene mutations have been associated to have a role in colon cancer and since gastric and colon tumors share some common genetic lesions, it is relevant to investigate the role of APC tumor suppressor gene in gastric cancer.MethodsWe investigated for somatic mutations in the Exons 14 and 15 of APC gene from 40 diffuse type gastric cancersamples. Rabbit polyclonal anti-APC antibody was used, which detects the wild-type APC protein and was recommended for detection of the respective protein in human tissues. Cell cycle analysis was done from tumor and adjacent normal tissue.ResultsAPC immunoreactivity showed positive expression of the protein in stages I, II, III and negative expression in Stages III and IV. Two novel deleterious variations (g.127576C > A, g.127583C > T) in exon 14 sequence were found to generate stop codon (Y622* and Q625*)in the tumor samples. Due to the generation of stop codon, the APC protein might be truncated and all the regulatory features could be lost which has led to the down-regulation of protein expression. Our results indicate that aneuploidy might occurdue to the codon 622 and 625 APC-driven gastric tumorigenesis, in agreement with our cell cycle analysis. The APC gene function in mitosis and chromosomal stability might be lost and G1 might be arrested with high quantity of DNA in the S phase. Six missense somatic mutations in tumor samples were detected in exon 15 A-B, twoof which showed pathological and disease causing effects based on SIFT, Polyphen2 and SNPs & GO score and were not previously reported in the literature or the public mutation databases.ConclusionThe two novel pathological somatic mutations (g.127576C > A, g.127583C > T) in exon 14 might be altering the protein expression leading to development of gastric cancer in the study population. Our study showed that mutations in the APC gene alter the protein expression and cell cycle regulation in diffuse type gastric adenocarcinoma.